伪狂犬病毒EP0基因的克隆及其在大肠杆菌中的表达研究  

Cloning of EP0 Gene of Pseudorabies Virus and its Expression in E.coli

作　　者：孙磊磊[1] 程艺[1] 梁梓森[1] 周慧英[1] 琚春梅[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《中国畜牧兽医》2012年第6期57-60,共4页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31001074);广东省科技计划项目(2008B020600003)

摘　　要：为了研究EP0蛋白在伪狂犬病毒粒子中的定位及其功能,试验利用PCR从伪狂犬病毒基因组中扩增PRV EP0基因的编码区,克隆至pMD18-T载体,构建重组质粒pMD-EP0。经测序鉴定正确后,用EcoRⅠ和HindⅢ双酶切pMD-EP0,回收EP0基因与经同样双酶切处理的pET-32a(+)进行连接,构建重组质粒pET-EP0。将pET-EP0转化大肠杆菌BL21(DE3),经IPTG诱导后,通过SDS-PAGE和Western blotting检测融合蛋白的表达情况及反应原性。结果表明,成功扩增得到EP0基因编码区序列,大小为1227bp;重组表达质粒pET-EP0经诱导后,EP0融合蛋白获得了表达,大小约为75ku,主要以包涵体形式存在,且能与伪狂犬病毒阳性血清反应。To study the location and function of EP0 protein in particles of pseudorabies virus, CDS region of EPO gene was amplified by PCR, and then ligated with pMD18-T to construct a recombinant plasmid pMD-EP0. After identification by se- quencing analysis, EPO gene was extracted from this plasmid digested by EcoR I and Hind Ⅲ, and cloned into pET-32a（＋） vector to construct a recombinant expression plasmid pET-EP0. This plamid was identified by PCR and sequencing analysis and then transformed into E. coli BL21（DE3）. The target protein was detected by SDS-PAGE and Western blotting. The result showed that CDS region of EPO gene, a fragment containing 1227 bp was amplified successfully and EP0 protein was expressed efficiently with the size of 75 ku and existed in inclusion body. The result of Western blotting showed that the expressed EP0 nrotein could be combined with L positive serum of PRV.

关 键 词：伪狂犬病毒 EP0基因 克隆 表达 

分 类 号：Q78[生物学—分子生物学]